John R. Thompson
jrt at home.com
Sun Mar 15 20:21:47 EST 1998
RMARTIN at INDY.TDSNET.COM (RICHARD MARTIN) wrote:
>I am contemplating an experiment involving ligation of contiguous
>oligonucleotides on a ssDNA template of about 500-1000 bases in length
>but I am unsure of several points. Can anyone help with input and
>literature references? Here are my questions:
>1. How easily can a string of oligos be ligated together?
>Specifically, does anybody have information on the processivity,
>efficiency, and fidelity of available ligases for ligated together
>multiple oligos in a single reaction? For example, can a string of
>more than 100 different 6-mers be faithfully ligated together into a
>600 bp fragment with reasonable yield?
>2. Also, how small can the oligos get and still be efficiently
>ligated together? Can 5-mers be used?
The low annealing temperature required for such small oligos would be
the main limitation I would think. Also I believe the oligos come off
the machine with 5' hydroxyls; you'll need to phosphorylate with
polynucleotide kinase before they will ligate.
Why use such short oligos? You pay by the base and oligos up in the
60mer range are fairly routine.
Brendan Cormack et al. used overlapping oligos to construct a Candida
codon-optimized GFP (~760bp) in a Microbiology paper (1997) (sorry
don't have the full ref handy).
Merck Research Labs
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